Plants are known to synthesize very long chain fatty acids (VLCFAs). VLCFAs are saturated or unsaturated monocarboxylic acids with an unbranched even-numbered carbon chain that is greater than 18 carbons in length. Many VLCFAs are 20–32 carbons in length, but VLCFAs can be up to 60 carbons in length. Important VLCFAs include erucic acid (22:1, i.e., a 22 carbon chain with one double bond), nervonic acid (24:1), behenic acid (22:0), and arachidic acid (20:0).
Plant seeds accumulate mostly 16- and 18-carbon fatty acids. VLCFAs are not desirable in edible oils. Oilseeds of the Crucifereae (e.g., rapeseed) and a few other plants, however, accumulate C20 and C22 fatty acids (FAs). Although plant breeders have developed rapeseed lines that have low levels of VLCFAs for edible oil purposes, even lower levels would be desirable. On the other hand, vegetable oils having elevated levels of VLCFAs are desirable for certain industrial uses, including uses as lubricants, fuels and as a feedstock for plastics, pharmaceuticals and cosmetics.
The biosynthesis of saturated fatty acids up to an 18-carbon chain occurs in the chloroplast. C2 units from acyl thioesters are linked sequentially, beginning with the condensation of acetyl Coenzyme A (CoA) and malonyl acyl carrier protein (ACP) to form a C4 acyl fatty acid. This condensation reaction is catalyzed by a β-ketoacyl synthase III (KASIII). β-ketoacyl moieties are also referred to as 3-ketoacyl moieties.
The enzyme β-ketoacyl synthase I (KASI) is involved in the addition of C2 groups to form the C6 to C16 saturated fatty acids. KASI catalyzes the stepwise condensation of a fatty acyl moiety (C4 to C14) with malonyl-ACP to produce a 3-ketoacyl-ACP product that is 2 carbons longer than the substrate. The last condensation reaction in the chloroplast, converting C16 to C18, is catalyzed by β-ketoacyl synthase II (KASII).
Each elongation cycle involves three additional enzymatic steps in addition to the condensation reaction as discussed above. Briefly, the β-ketoacyl condensation product is reduced to β-hydroxyacyl-ACP, dehydrated to the enoyl-ACP, and finally reduced to a fully reduced acyl-ACP. The fully reduced fatty acyl-ACP reaction product then serves as the substrate for the next cycle of elongation.
The C18 saturated fatty acid (stearic acid, 18:0) can be transported out of the chloroplast and converted to the monounsaturate C18:1 (oleic acid), and the polyunsaturates C18:2 (linoleic acid) and C18:3 (α-linolenic acid). C18:0 and C18:1 can also be elongated outside the chloroplast to form VLCFAs. The formation of VLCFAs involves the sequential condensation of two carbon groups from malonyl CoA with a C18:0 or C18:1 fatty acid substrate. Elongation of fatty acids longer than 18 carbons depends on the activity of a fatty acid elongase complex to carry out four separate enzyme reactions similar to those described above for fatty acid synthesis in the chloroplast. Fehling, Biochem. Biophys. Acta 1082:239–246 (1991). In plants, elongase complexes are distinct from fatty acid synthases since elongases are extraplastidial and membrane bound.
Mutations have been identified in an Arabidopsis gene associated with fatty acid elongation. This gene, designated the FAE1 gene, is involved in the condensation step of an elongation cycle. See, WO 96/13582, incorporated herein by reference. Plants carrying a mutation in FAE1 have significant decreases in the levels of VLCFAs in seeds. Genes associated with wax biosynthesis in jojoba have also been cloned and sequenced (WO 95/15387, incorporated herein by reference).
Very long chain fatty acids are key components of many biologically important compounds in animals, plants, and microorganisms. For example, in animals, the VLCFA arachidonic acid is a precursor to many prostaglandins. In plants VLCFAs are major constituents of triacylglycerols in many seed oils, are essential precursors for cuticular wax production, and are utilized in the synthesis of glycosylceramides, an important component of the plasma membrane.
Obtaining detailed information on the biochemistry of KAS enzymes has been hampered by the difficulties encountered when purifying membrane bound enzymes. Although elongase activities have been partially purified from a number of sources, or studied using cell fractions, the elucidation of the biochemistry of elongase complexes has been hampered by the complexity of the membrane fractions used as the enzyme source. For example, until recently, it was unclear as to whether plant elongase complexes were composed of a multifunctional polypeptide similar to the FAS found in animals and yeast, or if the complexes existed as discrete and dissociable enzymes similar to the FAS of plants and bacteria. Partial purification of an elongase KAS, immunoblot identification of the hydroxy acyl dehydrase, and the recent cloning of a KAS gene (FAE1) suggest that the enzyme activities of elongase complexes exist on individual enzymes.